Implant element

ABSTRACT

An implant element for permanent anchorage in bone tissue in which at least the surface is intended to face the tissue in the implantation region. The element is made of titanium with a titanium oxide surface which has been modified by anodization to acquire oxide thickness of approximately 10-200 nm. Also acquired are an increased surface crystallinity and a roughness on the submicrometer scale in order to provide a high-degree of bone-to-implant contact.

This application claims priority to PCT Application No. PCT/SE98/00891,filed May 14, 1998, and Swedish Application No. SE 9701872-5, filed May16, 1997.

BACKGROUND OF THE INVENTION

Oral implants are made of syntetic materials and inserted in mucosalsoft tissues and bone to serve as anchorage for prostheticconstructions. The choice of materials for bone anchorage has beendiscussed and considered over the years (reviewed in (Br{dot over(a)}nemark, 1996)) Osseointegrated titanium implants ad modum Br{dotover (a)}nemark have been successfully used for 30 years. There areseveral factors which are assumed to play important roles for theoutcome of this treatment, for instance the choice of titanium with itsadequate mechanical properties and corrosion resistance (Williams,1981), surface topography and the relatively non-traumatic surgicalprocedures.

It is assumed that two-stage surgical procedure with an earlypost-operative period (3-6 months) without loading is important for theinitial implant stability during the early healing phase. However, thetwo-stage surgical technique may be a disadvantage for the patient andrequires more resources. In clinical practice, not only the materialsand surgical procedures but also systemic and local host factors set thelimits for treatment. It has been found that the failure rates arehigher in the maxilla and the posterior mandible and that the successrates very much depend on the quality of bone (Esposito et al., 1997).It is therefore of importance to identify the beneficial and negativefactors related both to the implant and the host in order to optimizethe implant treatment. A reduction of the healing period and amaintenance of long-term stability during clinical loading conditionstherefore appears essential.

A biomaterial is a material used in a medical device, intended tointeract with biological systems (Black, 1992). The materials used inman-made structures may be divided into three classes: metals, ceramicsand polymers. The classes are distinguished by the type of interatomicbonding (Cooke et al., 1996).

Metals consist of a large number of small crystallites. Each crystalliteis an aggregate of atoms regularly arranged in a crystalline structure.When molten metals (which are amorphous) solidify small crystals(grains) start to grow. The irregularly arranged crystals eventuallymeet each other which gives rise to boundaries between the crystals,grain boundaries. The imperfect packing of atoms in the boundariesconstitutes weak points in the material, which will be most stronglyaffected by a surface treatment such as etching or plasma cleaning and agroove will be created showing up as a darker line. The surfaceproperties of a material is different from the bulk properties.

The term commercially pure (CP) titanium is applied to unalloyedtitanium and includes several grades containing minor amounts ofimpurity elements, such as carbon, iron and oxygen. The amount of oxygencan be controlled at different levels to provide increased strength.There are four grades of titanium where grade 1 (used in the presentthesis) contains the lowest amount of oxygen. The microstructure of CPtitanium is essentially all α titanium which has a HCP crystalstructure.

Titanium dioxide, TiO₂, is the most common and stable of the titaniumoxides, while Ti₂O₃ and TiO are more rare (Lausmaa, 1991).

TiO₂ can exist in three crystalline modifications; anatase (tetragonalstructure), rutile (tetragonal), and brookite (orthotrombic). Rutile andanatase are the most usual forms whereas brookite is very rare (Keesman,1966).

Techniques have been developed to alter and modify the surfaceproperties of implants via mechanical and chemical procedures (Lausmaa,1996; Smith et al., 1991a; Smith et al., 1991b). Plasma-spraying,sputter deposition, oxidation, vaporization, (grit, sand) blasting,grinding, etching, plasma cleaning and ion bombardment are examples oftechniques available for this purpose.

Electropolishing is an electrochemical technique often used to obtain animproved surface finish by controlled dissolution of the surface layerof the metal. The amorphous surface layer produced by the machining ofthe implants is removed. After electropolishing a polycrystallinesurface with a surface oxide consisting mainly of TiO₂, typically 3-5 nmthick as measured by X-ray photoelectron spectroscopy (XPS), is found onthe surface (Lausmaa, 1996).

Anodic oxidation is an electrochemical method used to increase thethickness of the oxide layer on metal implants. A current is applied inan electrolytic cell in which the sample is the anode. When a potentialis applied on the sample, the current will transport oxygen containinganions through the electrolyte and a continuous oxide is formed on themetal sample. The stoichiometry of anodic oxides on titanium is mostlyTiO₂. The anodic oxides on titanium contain various structural featuressuch as porosity (Lausmaa, 1996).

In order to characterize the surface properties after the modificationsthe following techniques were used; SEM and AFM for surface topographyand roughness; ESCA and AES for surface composition and oxide thickness.

Interactions Between Titanium Surfaces and Proteins/Cells/Tissues

A review of the literature shows that surface modifications influencethe biological response. The first events that take place when animplant is inserted in vivo is the exposure of the material surface towater and biomolecules, including plasma proteins. Both under in vitroand in vivo conditions serum proteins are known to adsorb to foreignmaterial surfaces within seconds. The adsorption and desorptionphenomena on different biomaterial surfaces have been studied intensely.A working hypothesis is that the biological response is directed by theinitial protein adsorption which subsequently influence thecellular/tissue response and ultimately the performance of the implant(Horbett, 1996).

Three types of adsorption/desorption patterns have been described formetals and their oxides (Williams and Williams, 1988). For example,titanium was found to adsorb low levels of albumin, which remained lowduring a 48 h period. In addition, the albumin desorbed relativelyeasily. Other metal surfaces such as vanadium, showed an initially lowamount of albumin, but the amount increased and desorption was slow.Gold was found to be characteristic for a surface with a high initialadsorption of albumin and the amount increased throughout theexperiment.

A modification and variation on surface properties and the resultingeffects on molecular adsorption to surfaces may provide importantinsights into the role of surface properties for biological reactions.Modified and characterized surfaces have been used to detect differencesin the behaviour and adsorption patterns of proteins (McAlarney et al.,1991; McAlarney et al., 1996; Nygren, 1996; Shelton et al., 1988; Sunnyand Sharma, 1990; Tengvall et al., 1992; Wälivaara et al., 1994;Wälivaara et al., 1992). Shelton et al (1988) found that a larger amountof proteins were adsorbed to negatively charged polymer beads than topositively charged beads but the roughness of the surface did not seemto influence protein adsorption or cellular behaviour. In general, roughsurfaces are considered more wettable than smooth surfaces which may bean effect caused by an increase of the surface area as well as by anincreased hydrophilicity of the surface (Curtis et al., 1983).

Nygren (1996) found two different reactions when hydrophilic andhydrophobic titanium surfaces were exposed to whole blood. On thehydrophobic surface, adherent platelets and fibrinogen were presentwhile complement factor 1 (C1) and prothrombin/thrombin were present onthe hydrophilic surface. Baier et al. (1982) has reviewed the principlesof adhesive phenomena in diverse systems and he pointed out thewettability of a surface as the important parameter influencing theprotein adsorption pattern.

The surface energy of a material is influenced by various cleaningprocedures and the oxide thickness. According to Sunny and Sharma (1990)an increase of the oxide layer on aluminium, increased thehydrophobicity of the surface, resulting in an increased adsorption offibrinogen. In addition, the glow discharge technique rendered thesurface more hydrophilic causing less fibrinogen adsorption. However,other results were obtained by Wälivaara et al (1994) who found that thetitanium oxide thickness and carbon contamination had no influence onprotein adsorption and contact activation. Interestingly, increasedsurface concentrations of complement factor 3 (C3) was correlated withan increasing titanium dioxide film thickness and/or crystallinity. Theoxide crystallinity seemed to be of more significance than the oxidethickness (McAlarney et al., 1996). In another study, McAlarney (1991)found that C3 adsorbed preferentially onto grain boundaries which may beexplained by the differences in surface energy between grain boundariesand bulk surface. It is known that titanium oxide surfaces bind cations,particulary polyvalent cations (Abe, 1982).

The oxide layer is highly polar and attracts water and water-solublemolecules. In general therefore, calcium ions may be attracted to theoxide surface by electrostatic interaction with oxygen (O⁻). In a studyby Lausmaa et al (1988), approximately 100 samples prepared according toclinical procedures were analyzed with ESCA. The spectra showed that thesurface consisted mainly of TiO₂. Carbon and smaller amounts of N, Cl,Ca, S, P, Na and Si were found on the surface but after sputtering allwere removed except for Ca which was found throughout the oxide.

It is of a major interest to understand, on a time-scale from immediateresponses to years, how material properties influence cellular activityin the interface and vice versa since rejection, excessive scarformation/encapsulation by fibrous tissue and restitution of originaltissue may largely influence the performance of the implant. In softtissues a fibrous capsule is formed around the implant (phenomenon ofwalling off the material from the biological environment) (Thomsen andEricson, 1991). In bone, encapsulation of the implant by fibrous tissuemay occur but is not obligatory and instead mineralized bone canestablish direct contact with the implant, a process calledosseointegration (Br{dot over (a)}nemark et al., 1969). Although thework on cell-material interactions has been intensified during recentyears, the mechanisms by which material properties influence biologicalreactions are still not clear. Studies in vitro.

The attachment of tissues to implants in vivo is a complex matterbecause in most cases there are different types of tissues involvedwhich may behave differently at different surfaces. The response ofcells to variations in culture substrate topography varies for differentcell types like macrophages (Rich and Harris, 1981; Salthouse, 1984),fibroblasts (van der Valk et al., 1983), periodontal cells (Cochran etal., 1994), epithelial cells (Chehroudi et al., 1989; Chehroudi et al.,1990), osteoblasts (Bowers et al., 1992; Martin et al., 1995) andchondrocytes (Schwartz et al., 1996).

Rich and Harris (1981) showed that macrophages accumulatedpreferentially on less hydrophilic as well as on roughened substrata.Murray et al (1989) showed that when macrophages adhered to hydrophilicsurfaces PGE₂ release and bone resorption was stimulated compared withhydrophobic surfaces. In addition, the rough surfaces was found tostimulate bone resorption to a greater extent than smooth surfaces.Although the roughness and the surface energy of the different surfaceswere not quantitated this indicates that the interactions betweenmacrophages and implant surfaces cause a release of factors which ishigher than if cells are in suspension. Studies on human monocyteinteractions with titanium surfaces have shown that the interleukin-1release by the cells is modulated by protein adsorption and the presenceof material particles (Gretzer et al., 1996).

Interestingly, different results have been obtained with fibroblasts.Human fibroblasts attached better to smooth than to rough titaniumsurfaces, (polished with 1 μm diamond paste versus the rougher; preparedwith 240 or 600 grit silicon carbide metallographic papers) (Keller etal., 1989). Spreading of fibroblasts was found to depend on the polarsurface free energy (van der Valk et al., 1983) since at least onvarious polymer surfaces, low cell spreading was found on low polarparts. Sukenik et al (1990) modified titanium surfaces with differentcovalently attached self-assembled monolayers (four different chemicalendgroups; CH₃; C═C; Br; Diol). The neuroblastoma cell attachment to thedifferent surfaces was comparable but cell spreading was leastpronounced on the most hydrophobic surface (CH₃ and C═C)

Osteoblasts are sensitive to subtle differences in surface roughness andsurface chemistry and respond to altered surface chemistry by alteringproliferation, extracellular matrix synthesis, and differentiation(Boyan et al., 1995). Osteoblasts exhibited different phenotypes whencultured on rutile or amorphous TiO₂ surfaces, but with the same oxidethickness and degree of roughness. Differences were therefore suggestedto be attributed to crystallinity alone (Boyan et al., 1995).

Osteoblasts have an initial greater attachment to rough, sandblastedtitanium surfaces with irregular morphology but average roughness(R_(a)) parameters did not predict cell attachment and spreading invitro (Bowers et al., 1992).

Proliferation and differentiation parameters in osteoblast-like cellswere modified by growing cells on titanium discs with an increasedroughness (15-18 μm) (Martin et al., 1995). Interestingly, cells atdifferent stages of differentiation responded differently to the samesurface (Boyan et al., 1995; Schwartz et al., 1996).

A basis for most studies in vitro on the role of surface properties forcell function is the adhesion of cells to the surface of the culturedish. The resulting interactions between the cell and the surface, withor without adsorbed molecules, is therefore a fundamental and obviouspart of the experimental set-up. In this context it may also be arguedthat the properties of the material surface as stimulating or inhibitingfactors on cells could be over-emphasized in relation to other potentialand maybe equally important modulating factors present in the vicinityof cells and surfaces in the complex biological situation in vivo.

Studies on titanium implants in bone (Sennerby et al., 1993a; Sennerbyet al., 1993b), indicated that osteoblasts did not adhere to the implantsurface and that formation of bone was not initiated at the surface.This observation suggests that the studies on the interaction betweenosteoblasts and titanium surfaces in vitro is of minor relevance.Nevertheless, studies in vitro, where various aspects of the complex invivo situation can be studied in detail may be of great value but thisrequires that the conditions in vivo are considered when the in vitrosystem is designed.

On the basis of the published in vitro studies it may be concluded thatthe surface roughness appears to influence the cell proliferation albeitdifferently depending on the degree of cell maturation. Differences insurface properties may influence the cell attachment and proliferationalthough the mechanism is not clear. It is also evident that differentcell types are differently influenced by the surface properties.However, so far there are few studies on the effect of modified titaniumsurfaces on cellular behaviour. A review of the literature on the invivo response to titanium implants is therefore appropriate.

In general, histology, histochemistry and immunohistochemical techniqueshave been used for the evaluation of soft tissue reactions. Due totechnical difficulties to obtain thin sections of an intact metal-tissueinterface the ultrastructure of the interface tissue has been difficultto study (Ericson and Thomsen, 1995). However, for metals in softtissues, an electropolishing method by which the bulk metal, but not thethin surface oxide layer, is removed (Bjursten, 1990) have made suchstudies possible.

The macrophage plays a pivotal role during healing of soft tissue aroundimplants. The soft tissue response to titanium implants in rats isdescribed by Thomsen and Ericson in (Br{dot over (a)}nemark et al.,1995). A fluid space, containing cells and proteins was present duringthe early phase (1-2 weeks) after introduction of a titanium implant insoft tissues (Johansson et al., 1992; Röstlund et al., 1990). Theconcentration of leukocytes and the proportion of PMN in the fluid spacedecreased between 1 and 7 d (Eriksson et al., 1994). After one week themajority of inflammatory cells in the fluid space, predominantlymonocytes and macrophages, were attached to the fibrin matrix at theborder between the fluid space and the reorganized tissue rather than tothe implant surface. After six weeks the fluid space was largely absentand the macrophages had established contact with the implant surface(Johansson et al., 1992; Röstlund et al., 1990). Macrophages constitutedthe most common cell type at the titanium surface, and exhibiteddifferent phenotypes, as judged by their ultrastructure (Johansson etal., 1992). Immunohistochemical observations (Rosengren et al., 1993)show that the fluid space around a titanium implant one week afterimplantation contained albumin, complement factor C3c, immunoglobulins,fibrinogen and fibronectin. Albumin and C3c were distributed in thefluid space and throughout the tissue interstitium during the firstweek. Fibrinogen and fibronectin co-localized preferentially at theborder between the fluid space and the tissue, thus forming aprovisional matrix to which macrophages and fibroblasts adhered.

After 6 and 12 weeks, fibrinogen was not detected in the surroundingtissue whereas strands of fibronectin was found in the surroundingcapsule (Rosengren et al., 1996). Collagen type I immunoreactivity,coinciding with the collagen bundles in the surrounding tissue, had adistribution similar to that of fibronectin, reaching close to thetitanium surface, but always separated from it by one to several layersof macrophages after 12 weeks.

The general sequence of cellular migration and accumulation as well asthe leakage of plasma into the tissue in the immediate vicinity of theimplant surface has been observed after implantation of severaldifferent materials, including metals, in soft tissues (Thomsen andEricson, 1991). The tissue response around nitrogen-ion implantedtitanium discs inserted in the rat abdominal wall of rats was notsignificantly different from that observed around pure titaniumimplants. However, after 6 weeks a predominance of macrophages andmultinuclear giant cells was found around the nitrogen-ion implanteddiscs (Röstlund, et al., 1990). A comparison of titanium and Ti6Al4Vafter 1, 6 and 12 weeks in the same rat abdominal wall model did notreveal any differences with regard to cell types and numbers in theinterface (Johansson et al., 1992). Further, the authors did not findany difference in fibrous capsule width. Therin et al. (1991) showedsimilar results when comparing the capsule thickness for titanium,TiO₂-coated titanium, Ti6Al4V, TiO₂-coated Ti6Al4V, TiN-coated Ti6Al4V,Ti5Al2.5Fe and stainless steel (316 L).

In contrast to polymers (Chehroudi et al., 1989; Chehroudi et al., 1990)studies in soft tissues which have been focused on the biologicaleffects of altered surface topography and roughness of metal implantsare relatively few.

However, in an extensive light microscopical study on the effects ofsurface roughness variations of titanium and stainless steel,(Ungersböck et al., 1994) it was shown that smooth implants induced athicker soft tissue capsule with an intervening fluid space. Incontrast, blasted and anodized titanium plates with relatively highvalues of roughness parameters (Ra 0.75) were surrounded by asignificantly thinner soft tissue layer without a continuous liquidspace. On the basis of these results it is difficult to conclude thatthere exists a simple relationship between increased surface roughnessand capsule thickness. For instance, Al₂O₃-blasted titanium plates withan even greater surface roughness (R_(a) 1.5) had a capsule thicknesswhich was similar to that around blasted, anodized titanium samples.Further, tumbled titanium plates (R_(a) 0.15) had a capsule thicknesswhich was similar to tumbled and anodized smooth titanium (R_(a) 0.33).The roughness was measured with a profilometer and the elementalcomposition of implant surfaces was not reported. It is thereforepossible that the surface chemical composition and/or roughness on thesubmicrometer level, differed between the samples. Studies on theeffects of various surface topographies (smooth vs. variousmicrotextures between 1 and 10 μm) of titanium discs implanted in softtissues of rabbits showed that collagen type III immunoreactivity wasdetected in the fibrous capsule around several materials, but thatcollagen type I was positively stained only in capsules around titanium(von Recum et al., 1993).

In general, the experimental studies in soft tissues indicate thatmetals become surrounded by a fibrous capsule with macrophages locatedclosest to the surface, thus separating fibroblasts from the surface. Sofar there are few available morphological data on the interfacestructure around titanium surface modifications. It is still an openquestion how the material surface properties influence proteinadsorption during in vivo conditions and how the surface propertiesinfluence the cells close to the surface. Moreover, it is not understoodhow the composition and structure of the surrounding fibrous capsule isinfluenced by the material surface-macrophage interactions. It is likelythat several additional factors must be considered, including leachingof metal ions, loading conditions and micromovements between the implantsurface and tissue.

The response of bone to injury is regeneration followed by remodellingof the newly formed bone in the direction of stresses. Analogously, whenan implant is inserted in bone, a similar cascade of events is expectedto occur including the recruitment of mesenchymal cells to the woundsite, their differentiation into osteoblasts, synthesis of osteoid, andcalcification of the extracellular matrix. The mesenchymal progenitorcells are pluripotent and able to differentiate into osteoblasts,chondrocytes, muscle cells and fat cells (Caplan and Boyan, 1994). Thepathway of differentiation of the mesenchymal cells as well asregeneration of bone around an implant is most likely dependent on acombination of factors including the degree of trauma, local andsystemic factors as well as implant properties and stability.

In the following a short summary of previous work on the interactionbetween metal implants and bone will be given. The performance ofnon-metal implants is reviewed elsewhere (de Groot et al., 1994).

Studies comparing the performance of, different implants of metalsincluding Vitallium®, niobium, titanium, titanium alloy, stainless steel(Johansson et al., 1991), and zirconium (Albrektsson et al., 1985;Johansson et al., 1994) in bone, did not reveal any major qualitativedifferences. The threaded titanium implants were in general found to bein contact with more mineralized bone than the other types of metal. Themechanisms for this is not clear nor is it understood why the propertiesof titanium are advantageous for biological applications compared withother metals, including those nearby in the periodic system. The goodbiological performance of titanium has been attributed to the titaniumoxide layer covering the surface, but no compelling evidence for thisview has been presented.

Several studies have indicated that an increased roughness of implantsurfaces (within a certain range) enhance the biomechanical performanceof implants. However, the bone response seldom show differences althoughsome studies indicate an increased bone-implant contact with increasedsurface roughness (Buser et al., 1991; Goldberg et al., 1995; Gotfredsenet al., 1995). Most studies did not reveal such a correlation (Carlssonet al., 1988; Gotfredsen et al., 1992; Thomas and Cook, 1985; Thomas etal., 1985; Thomas et al., 1987; Wennerberg 1996; Wilke et al., 1990;Wong et al., 1995). Br{dot over (a)}nemark (1996) made a correlationbetween morphological parameters of osseointegration of threadedtitanium implants and different biomechanical tests and found thatpull-out tests mainly reflects the mechanics of the surrounding bonewhile removal torque tests reflects the shearing forces leading toplastic deformation of the bone-implant interface. Possiblybiomechanical tests performed on implants with a rough surface(micrometer level), inserted in bone mainly reflect the bone-materialmechanical interaction (interlocking) although it cannot be excludedthat differences in the structure of the interface not resolved by lightmicroscopy are of importance.

Using an animal model similar to that used in the present study Sennerbyet al (1993b), studied the bone response 3-180 days after insertion ofscrew-shaped titanium implants. At 3 days mesenchymal cells weremigrating into the injury area around the implants. The implant surfacewas temporarily covered by multinuclear giant cells which disappearedwith time and when bone-titanium contact increased. Newly formed boneextended from the endosteal surface towards the implant and was alsoformed as islands within the implant threads.

With time the two types of newly formed bone fused. The threadsoriginally protruding into the marrow cavity were gradually filled withbone which matured by remodelling. Formation of new bone directly at thetitanium surface was not observed at any time interval.

Only a limited number of studies of the ultrastructure of the bone-metalinterface tissue are available. This may reflect the fact that thepreparation of the interface tissue for analysis by transmissionelectron microscopy TEM is technically demanding, especially when thedecalcification step is omitted.

Albrektsson et al (1982) introduced polycarbonate plugs coated with athin layer of evaporated metal as a model for metal implants. The plugswere implanted in the rabbit tibia. TEM on partially decalcifiedspecimens showed the presence (after 3 months) of collagen bundles closeto titanium implant but the last 100-500 nm closest to the implantconsisted of randomly arranged filaments. A 20-40 nm thick layer ofpartially calcified amorphous substance, suggested to consist ofproteoglycans was found in contact with the implant surface. A gradientof decreasing mineralization towards the implant surface was alsodescribed. In contrast, a larger number of macrophages and osteocyteswere found at gold-coated plugs. In more recent studies based on theplastic plug technique, other metal coatings including zirconium hasbeen compared with titanium (Albrektsson and Hansson 1986; Albrektssonet al., 1985).

Linder et al (1989), studied the interface morphology of plugs oftitanium. Ultrastructural observations in rabbit cortical bone (11months observation period) adjacent to titanium, Tivanium®, Vitallium®,and stainless steel revealed an unpredictable variation in interfaceultrastructure within 500-1000 nm of all metal surfaces. Three maintypes of interface structure were found; a) More or less regularlyarranged fibrils of collagen, with the longitudinal cross-banding of 68nm typical of type-I collagen, approaching the metal surface to within50 nm: b) Type-I collagen fibrils separated from the implant by a zoneof indistinct structures, but with some filamentous material, most oftenabout 500 nm in thickness, but sometimes up to 1000 nm; c) Type-Icollagen fibrils separated from the implant by a 500-600 nm zone of thinfilamentous structures, clearly more dense than in b. There was nostructural feature that was specific for a particular material (Linderet al., 1989).

Sennerby et al (1992) examined the interface morphology of titaniumimplants inserted into the rabbit tibia for 12 months and foundmineralized bone to be present very close to the implant surface withoutany apparent decreasing gradient of the concentration of bone mineraltowards the implant surface. A thin layer of amorphous non-mineralizedmaterial (100-200 nm wide) was present peripheral to the mineralizedbone. In addition, visible when mineralization was low, an about 100 nmwide electron dense lamina limitans was found to form the border betweenmineralized bone and the amorphous layer. This lamina limitans wereoften seen in direct continuity with lamina limitans bordering osteocytecanaliculi or separating bone of different mineralization grades.

Steflik studied the interface morphology at various types of implants inthe dog mandible using TEM and high voltage TEM and found an about 50 nmwide electron dense deposit at the implant surface (Steflik et al.,1992a; Steflik et al., 1992b). No difference was seen between loaded andunloaded implants (Steflik et al., 1993).Nanci et al (1994) studied thetissue response to titanium implants inserted for 1 day to 5 months intibia and femur of rats. The morphology of the interface tissue varied.Most often the interface between bone and the titanium implant consistedof a thin, electron-dense layer. This interfacial layer was found bothadjacent to mineralized bone and unmineralized collagen. Withimmunocytochemical techniques, the electron-dense layer described aslamina limitans was shown to be immunoreactive for osteopontin. Thecement lines in the surrounding bone often in continuity with the laminalimitans at the implant surface, showed a similar immunoreactivity forosteopontin. Osteocalcin, fibronectin, and albumin showed nopreferential accumulation at the interface. In a recent study McKee andNanci, (1996) are suggesting that osteopontin functions as a mediator ofcell-matrix and matrix-matrix/mineral adhesion during the formation,turnover and repair of mineralized tissue. A review of the literature onthe soft tissue response to titanium implants is important since apenetration through skin and mucous membranes is necessary to allow theattachment of external prosthetic appliances (e.g. teeth andepistheses). Interest has been focused on the prerequisites for anadequate adaptation of the soft tissue to the penetrating element.Empirically it has been found that a careful surgical technique withminimal motion at the interface by a tight adherence of the soft tissuesto the underlying bone may provide adequate conditions for clinicalpercutaneous and permucosal implants/anchorage units.

In studies on the relationship between the titanium surface andepithelium and connective tissues the majority of observations in humanshave been made in specimens from the oral cavity (Sanz et al., 1991;Seymour et al., 1989; Tonetti et al., 1993) and from the craniofacialregion (bone conductive hearing aids) reviewed in (Holgers 1994). In alight microscopic and ultrastructural study of oral implants (Sanz etal., 1991) the inflammatory infiltrates were scarce in the non-infectedperi-implant tissue. However, when gingivitis was observed, theinflammatory infiltrates were larger, dominated by mononuclear cells andplasma cells. (Seymour et al., 1989) characterized the mucosa aroundBr{dot over (a)}nemark osseointegrated titanium implants. The sampleswere obtained from healthy mucosa or with clinical signs of inflammation(gingivitis). The authors reported the presence of inflammation in bothsituations (healthy gingiva or gingivitis) but found larger inflammatoryinfiltrates and higher cell numbers when clinical signs of gingivitiswere present. The authors concluded that the mucosal reaction was astable and well controlled response. Similar findings were reportedaround clinically functioning bone-anchored percutaneous implants(Holgers, 1994), suggesting that an immunological compensation for theloss of barrier function is present at implants with clinicallyirritated skin. The relationship between epithelial cells and thesurface of implants as well as the common observations of epithelialdowngrowth have been suggested to play an important role for thefunction of implants, both in oral and percutaneous applications. Incontrast to observations for dental implants (Listgarten and Lai, 1975;Schroeder et al., 1981), no close contact between theepithelium/collagenous tissue and the surface of percutaneous titaniumimplants were seen (Holgers et al., 1995).

In conclusion, these observations indicate that machined titaniumimplants in soft tissues of humans are surrounded by inflammatory cellswhich appear to provide a protective barrier which may compensate for anon-optimal epithelial barrier.

Analysis of a retrieved osseointegrated clinical titanium implant (3months) (Lausmaa J. 1988) revealed an increased oxide thickness (byfactor 2-3) compared with an unimplanted sample. Similar in vivo oxidegrowth have been reported earlier. By the use of Auger electronspectroscopy, McQueen et al. (1982) observed that after 6 years in humanjaw bone, the original 50 Å thick oxide layer on titanium implantsurfaces had increased to a 2000 Å thick oxide layer.

Sundgren et al (1986) investigated the interface of bone-titanium andbone-stainless steel in humans and found that both the thickness and thenature of the oxide layers on the implant had changed during the time ofimplantation. Depending on the location, the thickness remainedunaffected (cortical bone) or increased with 3-4 times (bone marrow). Inboth cases, Ca and P were incorporated in the oxides. For titaniumimplants the oxidation process occurred over a longer time period(several years).

In a light microscopical study by Sennerby et al. (1991), sevenclinically stable (1-16 years) osseointegrated dental implants, wereanalyzed morphometrically. The major part of the implants were incontact with mineralized bone (56-85%), irrespective of observationperiod. Carlsson et al (1994) evaluated the tissue around implants withdifferent roughness inserted experimentally in arthritic knees. Blastedtitanium and hydroxyapatite-coated implants were in contact with bonewhereas smooth titanium implants often were surrounded by fibrous tissue.

Sennerby et al (1991), examined the structure of the interface aroundseven clinically stable dental implants (1-16 years) by morphometry. Inareas with mineralized bone close to the titanium surface, anon-mineralized amorphous layer was observed. An electron dense laminalimitans-like line was observed between the mineralized bone and the100-400 nm wide amorphous zone.

Ultrastructural observations were made on the metal-bone of interface ofimplants inserted in the tibia of patients with arthrosis and rheumatoidarthritis (7-20 months) (Serre et al., 1994) The implants were allscrew-shaped pure titanium implants and they were all “osseointegrated”.No difference between the ultrastructure of the interface between normalbone and implants compared with the interface of arthrotic and arthriticbone was observed. The heterogeneity of the interface was also confirmedin this study although the 100-400 nm wide amorphous zone reported bySennerby et al (1991), was not found.

In an ultrastructural study of the interface of a plasma-sprayedtitanium dental implant inserted in man (ITI), (Hemmerlé and Voegel,1996), two different interfacial structures were noticed. Both bonecrystals directly apposed on the implant surface and a granularelectron-dense substance interposed between the plasma-sprayed coatingand the bone were observed. Rohrer et al (1995) examined non-decalcifiedhistologic sections from 12 osseointegrated titanium plasma spray-coated(TPS) and TPS-treated with hydroxyapatite implants (IMTEC) from onepatient. All implants were successful and stable after 1 year when thesamples were retrieved. Both implant types were used with the samesuccess and no morphological differences were observed between the twoimplant types.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be better understood by reference to the DetailedDescription of the Invention when taken together with the attacheddrawings, wherein:

FIGS. 1a to 1 c is a scanning electron micrograph (SEM) representing anelectropolished and anodized implant;

FIG. 1d is a SEM representing a machined and anodized implant;

FIG. 2a is an atomic force micrograph (AFM) representation of a roughpart of an electropolished and anodized implant;

FIG. 2b is an AFM representation of a smooth part of an electropolishedand anodized implant;

FIG. 2c is an AFM representation of a machined and anodized implant;

FIG. 3 is a graphical representation of the total number of cells in theexudates for each of the corresponding surfaces: machined;electropolished; and electropolished and anodized;

FIG. 4 is a graphical representation of the total amount of DNAassociated with each of the corresponding surfaces: machined;electropolished; and electropolished and anodized;

FIG. 5 is a graphical representation of total bone contact (%) indifferent threads after a 6-week implantation time;

FIG. 6 is a graphical representation of total bone contact (%) indifferent threads after one-year implantation time.

The following invention is based on a comprehensive experimental studyusing differently modified titanium surfaces. In the following theexperimental procedures are summarized. Details may be found in athesis, (C. Larsson: The Interface between bone and metals withdifferent surface properties) including the following papers;

I. C. Larsson, P. Thomsen, J. Lausmaa, M. Rodahl, B. Kasemo and L. E.Ericson. Bone response to surface modified titanium implants. Studies onelectropolished implants with different oxide thicknesses andmorphology. Biomaterials 1994 (15) 13, 1062-1074

II. C. Larsson, P. Thomsen, B-O Aronsson, M. Rodahl, J. Lausmaa, B.Kasemo and L. E. Ericson. Bone response to surface modified titaniumimplants. Studies on the early tissue response to machined andelectropolished implants with different oxide thicknesses. Biomaterials1996 (17) 6, 605-616

III. C. Larsson, P. Thomsen and L. E. Ericson. The ultrastructure of theinterface zone between bone and surface modified titanium. (Inmanuscript)

IV. C. Larsson, P. Thomsen, B-O Aronsson, M. Rodahl, J. Lausmaa, B.Kasemo and L. E. Ericson. Bone response to surface modified titaniumimplants. Studies on the tissue response after one year to machined andelectropolished implants with different oxide thicknesses. Journal ofMaterials Science: Materials in Medicine, submitted

V. C. Larsson, P. Thomsen, J. Lausmaa, P. Tengvall, B. Wälivaara, M.Rodahl, B. Kasemo and L. E. Ericson. Bone response to surface modifiedtitanium implants. Studies on the early tissue response to differentsurface characteristics. (In manuscript)

Threaded screw-shaped implants were manufactured by machining of: puretitanium (grade I, 99.7%) (Permascand, Ljungaverk, Sweden)

The implant surfaces were modified with different preparation techniques(summarized in table I). Details of the different surface modificationsare found in the separate papers (I-V). All implants had a length of 4mm and a diameter of 3.75 mm.

Circular, disc-shaped implants (ø 10 mm, thickness 1.8 mm), weremanufactured by machining of a titanium rod (99.7%) (Permascand,Ljungaverk, Sweden). These implants were used for studies on proteinadsorption in vitro and inflammation in soft tissues.

The techniques for preparation and surface modification of the threetypes of implants (machined, electropolished, and electropolished plusanodized) used in the additional experiments a) and b), are described indetail in paper I and II.

During the electropolishing technique the sample is used as an anode inan electrochemical cell. By varying the electrolyte composition andprocess parameters (temperature, voltage and current) in the cell,various surface treatments can be carried out, including electrochemicalpolishing (electropolishing) or anodic oxidation (anodization).

The electropolishing technique which acts as a controlledelectrochemical dissolution of the surface (Landolt, 1987), was carriedout at 22.5 V in an electrolyte consisting of a mixture of 540-600 mlmethanol, 350 ml butanol and 60 ml perchloric acid held at −30° C. Eachsample was polished for 5 min. which is estimated to remove less than100 μm of material from surface. The electropolishing procedure wascarried out in order to produce a smooth, mirror-like surface finish. Italso has the effect of removing the plastically deformed amorphoussurface layer which results from machining of the material. Afterelectropolishing, the samples were carefully rinsed in methanol in orderto remove electrolyte residues.

Anodic oxidation (anodization) (Ross, 1975) was carried out at 80 V in aM acetic acid electrolyte at room temperature. This procedure produced avivid, greyish-purple colouration of the surface, due to lightinterference in the thick oxide that was formed. It is well establishedthat the oxide thickness is linearly dependent on the applied voltage,with a growth constant. α Å 2-3 nm/V for titanium, depending onexperimental conditions. The anodized samples were carefully rinsed indeionized water followed by a rinse in ethanol.

Scanning Auger electron spectroscopy (AES; Perkin-Elmer PHI 660, EdenPrairie, USA) was used to analyze the surface elemental composition.Oxide thickness was estimated from AES depth profile analysis. At least2 different spots (ø 100 μm), located at the threaded part of the samplewere analyzed. Depth profiles were obtained at 2 points (ø 10 μm).

AES survey spectra were acquired from two areas of ø 200 μm on onesample of disc-shaped machined, electropolished and electropolished plusanodized titanium implants.

Scanning electron microscopy (SEM; JEOL JSM-T-300, and Zeiss DSM 982Gemini) was used to obtain an overall picture of the surface topography.Atomic force microscopy (AFM; Nanoscope III, Digital Instruments, USA)was used for a more detailed characterization of the surface topographyand roughness. The surface roughness (R_(rms)) and surface areaenlargement (A_(diff)) were calculated using the computer software ofthe AFM instrument.

Contact angles were measured using a Ramé-Hart goniometer, model 100.Advancing and receding contact angles were determined for titanium(control), electropolished and electropolished plus anodized samples,both with Millipore filtered water and with methylene iodide. One drop(5 μl) of the liquid, was placed on three different spots on eachsample. Both right and left angles of the drop were estimated and themean values calculated. The samples were cleaned (in addition to theconventional cleaning steps with trichlorethylene, acetone, and ethanol)with 95% ethanol and air-dried within 30 min prior to analysis. Surfaceenergy was calculated and preferred values of surface tension for theliquids in room temperature (Wu, 1982) were used for water and methyleneiodide.

The implants were ultrasonically cleaned in trichlorethylene; acetone;methanol. After surface modification (electropolishing and/or anodicoxidation), all implants received a final ultrasonic cleaning step inethanol (70%). Finally, implants were either autoclaved in 120° C. for15 min. or γ-irradiated at 28.9 kGy for 25 h at 30° C. The hydrogenperoxide treated implants were treated with 10 mM H₂O₂ for 40 h at 8° C.after the ultrasonically cleaning procedure. No additional sterilizationwas performed.

The disc-shaped implants were ultrasonically cleaned intrichlorethylene; acetone; ethanol. After surface modification(electropolishing and/or anodic oxidation), all implants received afinal ultrasonic cleaning step in ethanol.

Rat plasma was obtained from two rats and used in protein adsorptionexperiments.

Fifteen Sprague-Dawley rats, weighing about 250 g, were used for studieson cell recruitment and adhesion to titanium surfaces in soft tissues.

The surgery was performed according to procedures described earlier,(paper I-V) and implantation was made bilaterally in the proximal tibialbones. After incision through the skin and periosteum, a flap was raisedto expose the bone area. Thus, each animal received one implant of eachtype, respectively.

After the animals were killed, the implants and surrounding tissue wereremoved en bloc, further immersed in glutaraldehyde over night and thenpostfixed in 1% osmium tetroxide, for two hours. After dehydration theundecalcified specimens were embedded in LR White® (The London Resin CoLtd, Hampshire, England).

In studies on protein adsorption in vitro surface adsorbed proteins werecollected, separated (SDS-PAGE) and visualized (Western blot). In brief,discs were kept in 99.5% ethanol, ultrasonicated in 99.5% ethanol,washed three times and kept in 99.5% ethanol until use. Beforeincubation with proteins, the samples were placed in sterile-filteredHBSS with calcium. Rat plasma was incubated on three surfaces of eachkind (machined, electropolished and electropolished plus anodicallyoxidized titanium) during 1 min. at 37° C. Thereafter loosely attachedproteins were rinsed off and the surface adsorbed proteins removed bythe detergent SDS (2%) together with enzyme inhibitory agents. The totalamount of collected proteins was analyzed with BCA Protein Assay Reagent(Pierce, USA) using spectrophotometry (562 nm) and with rat albumin asstandard. Gel electrophoresis with precasted Tris-glycine (4-15%)gradient acryl amide gels (BioRad, Miniprotean II) was performed toseparate the proteins. After separation the proteins were transferred tonitrocellulose membranes (70 V, 3 h, Tris-glycine-SDS buffer) followedby blocking of unspecific antibody binding by incubation in 3% gelatinin Tris-NaCl buffer, pH 7.5. To detect specific proteins on the membrane3 incubation steps were performed (60 min., room temp.) inTris-NaCl-Tween buffer.

The primary step included rabbit anti-rat fibronectin (FN); goatanti-rat fibrinogen (fractions) (FBN); sheep-anti-rat albumin (Alb);sheep anti-rat immunoglobulin G (IgG). The secondary step includedbiotinylated donkey anti-sheep IgG; biotinylated goat anti-rabbit IgGand the tertiary step included streptavidin conjugated to alkalinephosphatase in Tris-NaCl-Tween (TTBS) buffer. Visualization of thelabelled proteins (samples and standard) was made by incubation inBCIP/NBT.

The implantation of implants in soft tissues was performed according topreviously described procedures (Lindblad M. et al., 1997). In brief, 15Sprague-Dawley rats, weighing about 250 g, were anesthetized with an i.pinjection (0.1 ml/100 g b.wt.) of a 1:1:2 solution of sodiumpentobarbital (Apoteksbolaget, Sweden; 60 mg/ml), 0.9% saline anddiazepam (Apozepam®, Apothekarnes Laboratorium AS, Norway; 5 mg/ml). Therats were shaved on the dorsum and cleaned with 2% Jodopax® in ethanol.Incisions, about 15 mm long and 10 mm apart were made in the dorsal skinalong the midline. Subcutaneous pockets were created by blunt dissectionand implant discs were placed in the pockets using a pair of titaniumtweezers. In six rats three incisions were made along each side of themidline. These rats received two of each type of implant: one of thesetwo implants was rinsed in sterile HBSS buffer (Hanks balanced saltsolution with CaCl₂ 2.9 g/l, pH 7.4) whereas the other implant wasrinsed in sterile saline. The other nine each received three implants,one of each type, which were rinsed in saline prior to insertion. Theskin was closed with non-resorbable sutures. After 1 (n=6), 3 (n=6) and7 (n=3) days the dorsal skin of anaesthetized rats was cleaned and therats killed by an i.p. overdose of pentobarbital. The sutures were takenaway and the wound surfaces were gently drawn apart with tweezers. Theimplants were removed and placed in a sterile polystyrene tissue culturedishes, containing 500 μl sterile HBSS (with calcium) and kept on ice.The remaining content (exudate) of the cavity was collected by rinsing,using repeated aspirations (5 times) of 500 μl (total volume) sterileHBSS (with calcium).

Each retrieved exudate was kept on ice until the determination of cellcount and cell types. The exudates were stained with Turk solution andthe proportion of different cell types was determined. The total mean ofthe number of cells found in the exudate was calculated from six (1 and3 d) and three (7 d) rats per time period and the percentage mean valuesof different cell types were calculated in the same way.

The determination of the amount of DNA associated with the implantsurfaces was performed with a fluorescence assay (Labarca C. and PaigenK. 1980). In brief, after the retrieval procedure each implant was putin 500 μl 5×10⁻² M sodium phosphate buffer with 2×10⁻³ M EDTA and 2 MNaCl. Thereafter, the implants were frozen at −20° C. After thawing andultrasonication of the cells on the implants (about 15 s at each side ofthe implant), 200 μl of the solution was added to 5×10⁻² M sodiumphosphate buffer with 2 M NaCl, supplemented with 1 μg ml⁻¹ of thefluorescence marker Hoechst 33258 (Sigma, USA) at room temperature(15-30 min.). The samples were measured in a luminescence spectrometerwith excitation and emission wavelengths of 360 and 450 nm,respectively. The total amount of DNA was determined from standardcurves (0.025-2.5 μg DNA per ml). The total mean of the DNA amountassociated with the implant surfaces was calculated from six (1 and 3 d)and three (7 d) rats per time period.

Ground sections of 10-15 μm thickness were prepared from implant/bonespecimens (Donath and Breuner, 1982), and examined, using a LeitzMicrovid equipment connected to a personal computer. Measurements wereperformed directly in the microscope. The contact ratio between theimplant surface and bone tissue was calculated. Similarly, theproportion of bone tissue within the threads along the implant wascalculated. The data are given as percentage bone in direct contact withthe implant (referred to as bone contact) and percentage of the totalarea within the threads occupied by mineralized bone (referred to asbone area). All five consecutive threads (with number 1 and 2 located inthe cortex) were evaluated. In the one-year study, the values for the 3best consecutive threads were also presented. The mean value for eachimplant type at each time period was calculated and compared. Afterpolymerization embedded implants were divided longitudinally by sawing.One half was used to prepare ground sections (Donath and Breuner, 1982)which were used for morphometric analysis (papers I-II, IV-V) asdescribed above. The other half was used for the preparation of sectionsfor light and electron microscopy (papers I, IV). The implant wascarefully separated from the plastic embedded tissue (Sennerby et al.,1992; Thomsen and Ericson, 1985). The cavity formed after implantremoval was filled with plastic resin and polymerized before sectionsfor LM (approximately 1 μm thick) were cut with glass knives. In thesesections appropriate areas were selected for ultramicrotomy. Ultrathinsections, contrasted with uranyl acetate and lead citrate were examinedin Philips EM 400 or Zeiss CEM 902 electron microscopes.

Results

A summary of the results from surface characterization of the differentsamples (papers I-V) is presented in Table I.

Machined (control), electropolished, electropolished plus anodized (21nm thick oxide) and electropolished plus anodized (180 nm thick oxide)were used in paper I and III. The machined titanium implants (control)had typical machining grooves in the width of 1-10 μm. Theelectropolished implants had a very smooth, mirror-like surface with noapparent surface features at low or high magnification. The anodized (21nm) samples also appeared smooth whereas the anodized (180 nm) hadporous regions irregularly distributed over the smooth surface whichmade the implant surface roughness heterogeneous (1 μm scale). R_(rms)values obtained by AFM are presented in Table I.

Machined (control), machined plus anodized (180 nm thick oxide)electropolished, and electropolished plus anodized (180 nm thick oxide)were used in paper I and III.

The machined (control) and machined plus anodized surfaces had a similarsurface appearance with typical machining grooves in the width of 1-10μm. The machined plus anodized surface also showed an additional,irregular surface roughness on the submicron level. The electropolishedsurface appeared very smooth whereas the electropolished plus anodizedsurface presented a heterogeneous surface with irregularly distributedsmooth and rough (10-100 μm large) areas. No machining grooves werevisible on the surfaces of the two groups of implants that had beenelectropolished. R_(rms) values obtained by AFM are presented in TableI. Scanning electron micrographs are shown in FIG. 1(electropolished+anodized implant, a-c; machined+anodized implant, d).AFM images are shown in FIG. 2 (electropolished+anodized implant a=roughpart, b=smooth part; machined+anodized implant, c).

Paper V:

In this paper machined, glow discharge cleaned and thermally oxidized,glow discharge cleaned and nitrided and hydrogen peroxide treatedimplants were used. The machined (control) surface had thecharacteristic grooves, 1-10 μm in width as described above. The twogroups which were plasma cleaned and subsequently oxidized and nitrided,respectively, had similar surface topography. The underlying grainstructure could be seen although grooves from the machining procedurewere clearly visible. The hydrogen peroxide treated implants showedclear traces from the machining procedure and had a woolly surface whichreflects the etching action of the treatment.

Surface composition and thickness of surface layers

The results of the AES analyses are summarized in Table I.

Papers I, III

All samples had a relatively consistent surface composition independentof preparation. All spectra were dominated by strong Ti, O and C signalsand trace amounts of Ca, S, Si, P, Cl and Na were detected. Ca and Sappeared more frequently on the control samples than on theelectrochemically prepared ones. Lower levels of C and othercontaminants were found on the anodized (80V) samples. The depth profileanalysis resulted in oxide thicknesses of 4, 4-5, 21, and 180 nm for thecontrol, electropolished, electropolished/anodized (10V) andelectropolished/anodized (80V) implants, respectively.

Papers II, IV

Irrespective of surface preparation all samples had a relatively similarsurface composition with strong Ti, O and C signals in the spectra . Thecarbon contamination varied between the different samples from Å 34 at %for the machined and machined-anodized samples to Å 25 at % for the twoelectropolished samples. Trace amounts Ca, S, P, Si were detected (fewpercent).

Paper V

On all samples Ti, O or N/O and C were the dominant elements. Allsamples showed relatively low levels (10-15 at %) of carboncontamination on the surfaces compared to other studies (typically 30 at% or more) (Lausmaa J. 1996).

The oxides on the control, glow-discharge oxide and H₂O₂ treated samplesrespectively, were nearly stoichiometric titanium dioxide and of similarthickness (4-7 nm).

In Additional Experiments:

The disc-shaped implants used for studies on protein adsorption andinflammation in soft tissues consisted of a TiO₂ surface oxide coveredby varying amounts of hydrocarbons and other trace impurities. For themachined control sample, carbon levels around 50%, and around 4% Ca andminor traces of S and P were detected. For the electropolished samplearound 30% C was detected, and traces of Ca, S, and Cl. Theelectropolished plus anodized sample had carbon levels around 20%, andaround 6% Ca and traces of S, Cl, Si and Fe. Except for the variationsin carbon levels, the disc-shaped samples had a similar surfacecomposition as the corresponding surfaces of threaded implants used inthe previous studies (paper I-V).

Contact Angles and Surface Energy

The contact angles were measured on the circular disc-shaped implantssince it is not possible to measure the contact angle on a screw-shapedimplant (Table II and III).

The contact angle (H₂O advancing) was lower for the electropolished plusanodized implant than for the machined and electropolished samples. Dueto porosities of the electropolished plus anodized surface, capillaryforces may spread the water, thus giving a lower water contact angle(Andrade, 1985). The electropolished plus anodized surface had thegreatest hysteresis (difference between the advancing and recedingangle) when measured with methylene iodide. Increased surface roughnessand differences in topography may lead to increased hysteresis. Sinceall surfaces have a similar chemical composition, all surfaces will havethe same “real” contact angle although the surface topography willinfluence the measured value.

The Tissue Response

Protein Adsorption in vitro

Our observations show that there are only small differences in theprotein adsorption pattern between the machined, electropolished andelectropolished plus anodized titanium surfaces. The total amount ofadsorbed plasma proteins was similar on the three surfaces. The proteinconcentrations obtained were for machined titanium 1.15 mg/ml,electropolished titanium 1.05 mg/ml and electropolished plus anodizeddiscs 1.25 mg/ml, respectively. Further, the content of selected plasmaproteins, albumin, fibrinogen, fibronectin and IgG was similar.

Inflammatory Reaction in Soft Tissue.

The total number of cells in the exudates and associated with thesurfaces of the machined, electropolished and electropolished plusanodized discs after different times of implantation are shown in FIGS.3 and 4, respectively. The number of cells decreased with increasingobservation periods at all implant types. No major differences inabsolute total cell numbers were detected between the surfaces. Machinedtitanium pre-incubated in saline was one major exception revealing thehighest cell numbers among all samples.

With one major exception, mononuclear cells (monocytes/macrophages,lymphocytes) were predominant in the exudates around the implants at alltime periods (1 d: machined Ti 33% (HBSS) and 33% (saline),electropolished 47% (HBSS) and 50% (saline, electropolished plusanodized (47% (HBSS) and 55% (saline; 3 d: machined Ti 81% (saline),electropolished 87% (saline), electropolished plus anodized 82%(saline); 7 d: machined Ti 89% (saline), electropolished 100% (saline),electropolished plus anodized 94% (saline). In contrast to the otherimplants, machined titanium had a markedly lower proportion ofmononuclear cells after 1 d, and a correspondingly higher proportion ofPMN. This discrepancy was not observed at other time points. Nodifference in the proportion of cells in the exudate was observedbetween implants which had been incubated in HBSS or saline.

Morphology and Morphometry

Light Microscopic Observation

Paper I

After 7 weeks, immature bone with a wowen character filled the corticalthreads around all implants. At this time period, the merelyelectropolished implants had less endosteal intramedullary downgrowth ofthe bone than the machined and the electropolished plus anodized (180 nmthick oxide) implants. The electropolished plus anodized implants hadthe highest bone contact, 50% versus 20%, for the merelyelectropolished. After 12 weeks, the general organization of the bonearound the implants was the same as that observed after 7 weeks. only asmall increase of the bone contact between 7 and 12 weeks were found forthe electropolished plus anodized implants, however, for the twoelectropolished samples with a thin oxide the increased bone contact hadreached the same level as the electropolished sample with thick oxide.

Papers II, IV

After 1 week the cortical bone was in general in close contact with themachined and machined/anodized implant types. Both the electropolishedimplant types had lower values for bone-implant contact at this timeperiod (<5%).

At 3 weeks newly formed bone from the endosteum reached the implant andfilled the threads which were initially protruding into the marrowcavity. No quantitative differences were detected between the groups.

At 6 weeks, the electropolished implant had a lower bone contact thanthe electropolished plus anodized implant as well as the machinedimplants FIG. 5. The electropolished implants also showed the lowestamount of bone within the threads. The two types of machined implantswere surrounded by wider bone collar than the electropolished implants.In general, the bone was to a large extent in direct contact with theimplant surfaces.

After 1 year, almost all threads were filled with lamellar bone and theimplant surfaces were in close contact with the surrounding bone(60-70%) (FIG. 6) The bone response to the different implant types weresimilar. The bone response around the electropolished implant group wasequivalent with the other groups after one year.

Paper V

After 1 week the cortical bone was often in close contact with theimplants although no new bone formation was seen at the cut edge of thecortex. No quantitative differences were seen between the groups.

At 3 weeks newly formed bone from the endosteum reached the implant andfilled the threads which were initially occupied with marrow tissue.Marked signs of resorption and osteoid characterized the surface of thecortical and trabecular bone facing the implant surface. Also at thistime point no differences in bone contact or bone area were detectedbetween the groups.

At 6 weeks, no qualitative or quantitative differences were foundbetween the groups. The bone was to a large extent in direct contactwith the implant surfaces and these observations were similar to thosedetected in paper II.

Ultrastructural Observations

Paper III

A generally low degree of mineralization was found around theelectropolished implants and together with the smooth fracture outlinethe production of ultrathin sections as well as the interpretation ofthe interface was made easier. There was constantly a layer of amorphousmaterial between the mineralized bone and the implant surface (0.2 μmwide). The electropolished plus anodized implants (thick oxide) weremore difficult to examine (similar for the machined implants) since aseparation of the implant from the plastic embedded tissue oftenresulted in disruption of the interface tissue. The bone around theelectropolished plus anodized implants had in general a much highercontent of bone mineral than the merely electropolished implants. Anamorphous layer was also found around these implants, generally somewhatthicker than for the merely electropolished implants. The presence of alamina limitans forming the border of the mineralized bone towards theimplant was usually found around the electropolished plus anodizedimplants with thick oxide.

Discussion

The relative importance of different surface properties for thebiological performance of implanted biomaterials is largely unknown. Thestrategy chosen in the work was to systematically change the surfaceproperties of titanium implants and then evulate the biological responsein an animal model.

The surface chemistry, topography and microstructure of titaniumsurfaces have been varied in a well controlled manner in this thesis.There are several ways to modify the different properties althoughkeeping all the parameters controlled is difficult.

Biocompatibility and Modified Titanium Surfaces

Implantation of a non-biological material in biological surroundingsleads to a time- and partly material-dependent sequence of inflammatoryand reparative processes, although, as reviewed in the Introduction, thevarious material-related factors that influence these responses are notfully understood it is evident that protein adsorption and cellularadhesion to material surfaces are essential components of the tissueresponses. In previous studies in this laboratory (reviewed in theIntroduction), the soft tissue reactions around machined titaniumsurfaces have been characterized, including the cellular distributionand structure of the titanium-metal interface in experimental and humanapplications.

In experimental studies in vivo and in different in vitro models, thesurface wettability, chemical composition, pattern of protein adsorptionand the influence of exogenous stimuli have been found to influence theinflammatory cell recruitment, distribution and secretory response.Further, on the background of an early (and transient) distribution ofmononuclear and multinuclear cells on the machined titanium surfaceduring the inflammatory events which precede bone formation in theinterface (Sennerby et al., 1993a; Sennerby et al., 1993b) studies onprotein adsorption and cellular recruitment and adhesion tosurface-modified titanium implants were initiated.

The observation from the present experiment on in vitro plasma proteinadsorption and cellular recruitment and adhesion in soft tissues of therat indicate that no major differences were observed at a few selectedtime points, irrespective of the different surface properties exhibitedby the machined, electropolished and electropolished titanium discs. Wehave no explanation for the relatively higher cell numbers on machinedtitanium after 1 day. In agreement with other recent observations(Thomsen et al., in manuscript), the machined titanium samples wereassociated with both a relatively greater influx and association ofcells to the surfaces after 1 day then the other materials. Further, ourdata indicate that this inflammatory response is higher if implants arepre-incubated in saline than HBSS (with calcium). Moreover, theinflammatory exudate around the machined implants was associated with amarkedly higher proportion of PMN. Interestingly, gold implants havingless inflammatory cells in the exudate then hydroxylated and methylatedgold, was associated with a similar, relatively greater PMN predominanceafter 1 day (Lindblad et al., 1997).

The present result together with previous experimental and clinicalstudies using machine implants in soft tissues, provide an indicationthat also the electropolished and electropolished plus anodized implantsbelong to a group of materials with soft tissue biocompatibleproperties.

The Osseointegration Process

The Osseointegration Process and Modified Titanium Surfaces

The systematic approach in papers I-V was undertaken with the purpose toevaluate if and how variations of the metal implant surface propertiescould induce a variation of the bone reactions, as evaluated by lightmicroscopic morphometry and ultrastructural analysis. Thus, thethickness, morphology, topography and chemical composition of thesurface layer could be more or less intentionally varied. Since themachined titanium constituted the implant on which surface modificationswere made and, further, since a volume of scientific data exists on thematerial, biological and clinical properties of machined titaniumimplants, these implants were always included as a reference in theseparate experiments.

In spite of the widespread use of machined titanium implants in bone,the mechanisms for achieving osseointegration has been less wellunderstood. Previous experimental studies using the same experimentalmodel as in this thesis (Sennerby et al., 1993a; Sennerby et al., 1993b)that the implant surface did not serve as an attachment for osteoblastsand no evidence was obtained indicating that mineralization wasinitiated on the surface: instead bone formation was observed after 3days in the endosteum from which bone trabeculae projected towards theimplant, and after 7 days as solitary islands within the threads. Inboth locations mineralization occurred by deposition of mineral in thecollagenous matrix. Thus, the bone was growing towards the implantsurface and the collageneous matrix of the interface zone was the lastpart of the surrounding bone to become mineralized. After longer timeperiods, observations from animal experiments and human retrievalstudies (reviewed in the Introduction) indicate that osseointegration ofnon-functionally and functionally loaded machined, threaded titaniumimplants is characterized morphologically by a high amount of remodeledbone within the threads, a high bone-implant contact and a separation ofmineralized bone from the implant surface by a thin zone of amorphousmaterial.

In summary, the surface modified titanium implants evaluated in thepresent study (paper I-V) were found to essentially share biologicalproperties with machined titanium: early bone formation proceededtowards the implant surface and at later time periods all implants wereosseointegrated.

A major exception was the relatively low bone contact observed withelectropolished implants in the early phase (papers I-II). A possibleexplanation for this observation could be the existence of an initial,larger gap between the electropolished implants and the surroundingtissue (due to the removal of less than 100 μm (Å50 μm) of the implantsurface during the electrochemical process). However, the lower rate ofbone formation around merely electropolished samples after 6 weeks incomparison with the electropolished plus anodized samples can not beexplained by the difference in possible initial gap between the implantsurface and the tissue. It is suggested that the combination of aheterogeneous submicron roughness (smooth/rough; 75%/25%), increasedoxide thickness (180 nm) and thereby an increased crystallinity on theelectropolished plus anodized surface are advantageous propertiesassociated with the electropolished plus anodized implants. Thiscombination of properties has not been utilized previosly as part of animplant element but reports in the litterature indicate that forinstance the degree of crystallinity may (as a single property) affectcell behaviour.

In studies in vitro an increased crystallinity (while keeping oxidethickness and roughness parameters constant) was found to influence thephenotypic expression of osteoblasts (Boyan et al., 1995). In vitrostudies have also shown that the roughness of the culture substratuminfluences osteoblast-like cell proliferation, differentiation andmatrix production (Martin et al., 1995). Further, cells at differentstages of differentiation in vitro respond differently to the samesurface (Boyan et al., 1995; Schwartz et al., 1996). Therefore, ifextrapolating to in vivo conditions it is possible that thetitanium-bone interactions could be different at early and late timeperiods depending on time-dependent changes in the interface of thetypes of cells present and their maturity stage.

Although conflicting data exist in the literature, previous studies invivo indicate that an increased surface roughness (on the >1 μm level)may promote bone adaptation to titanium surfaces (Buser et al., 1991;Goldberg et al., 1995; Gotfredsen et al., 1995). It is thereforeinteresting, on the basis of the present one year data (paper IV) thatfirstly, all surfaces (machined, machined plus anodized,electropolished, electropolished plus anodized), being relatively smoothcompared to sandblasted or plasma sprayed surfaces exhibited a highdegree of bone-to-implant contact and a high proportion of bone withinthreads, and secondly, that the morphometric values were equal to orhigher than the values given for relatively rougher surfaces in anotherstudy using the same experimental model (Wennerberg, 1996). We have noclear explanation for these findings. One possibility is that the smoothelectropolished surface had acquired a thicker oxide and thereby achanged topography during the longer implantation period (1 year). Someevidence that such processes may be operative is the finding, in humanretrieval studies, that the thickness of the oxide had increased withtime (Lausmaa, 1988; McQueen et al., 1982; Sundgren et al., 1986).Another possibility is that the rate of bone formation andmineralization around the machined and surface modified titaniumimplants was influenced by ion release. Titanium ions (Ti⁴⁺) have a doserelated inhibitory effect on calcification in vitro (Blumenthal andCosma 1989). The ion release rates in vitro from titanium materialsdecay with time due to self passivation (Healey and Ducheyne, 1992a;Healey and Ducheyne, 1992b). Therefore, due to a relatively thinneroxide we cannot exclude that the electropolished implants are associatedwith a higher ion release.

Another hypothesis is that the titanium surface oxide, through itsability to bind calcium could favour mineralization, which in turn mightbe beneficial for bone formation (Hanawa, 1990). However, it has notbeen shown that this would have an effect on osteoblast adhesion,proliferation, secretion of extracellular matrix and mineralization ofthe titanium-interface zone. Previous in vivo data (Sennerby et al.,1993a; Sennerby et al., 1993b) and the present data (papers I-V ) do notindicate that this is valid under the “vivo” conditions.

However, the anionic TiO₂ attracts cations, like for instance calcium,and it has been suggested that calcium binding may be one mechanism bywhich proteins adsorb to TiO₂ (similar to hydroxyapatite) (Ellingsen,1991). Pre-treatment of TiO₂ by adsorption of lanthanum ions causes anincreased adsorption of proteins, coinciding with an inferior boneresponse in rats and rabbits (Ellingsen and Pinholt, 1995). In addition,pre-treatment of titanium implants with fluoride ions has been shown toincrease push-out values (Ellingsen, 1995). Thus, a chemicalmodification of the titanium surface may influence the bone tissueresponse, possibly by the adsorption of proteins to the surface. Thishypothesis is mainly supported by in vitro studies which have shown thatchondroitin-4-sulphate is bound to TiO₂ in the presence of calcium ions.Thus, tentatively in the amorphous zone, calcium bound to TiO₂ couldpromote the adsorption of sulphated glycosaminoglycans (Collis andEmbery, 1992).

Taken together, chemical modifications of the titanium oxide surfacehave been found to affect the adsorption of macromolecules on thesurface, and the tissue response. There are yet no evidence, however,that the positive effects on the bone response are due to a process ofbone formation and mineralization which is directed outwards from theTiO₂-surface. A future approach with the purpose to further modulate thebone response may be to selectively adsorb/incorporate molecules to thesurface which could influence bone precursor cells/osteoblasts andenhance mineralization. However, since the interactions betweenproteins, cells and such a chemically treated surface may be influencednot only by the chemical properties of the surface but also by thesurface submicron roughness, an optimization of both chemical andsurface roughness parameters have to be concidered when new implants aredesigned. The present invention is not limited to be used as an implantsurface as such but may be utilized as a substrata for such purposes.

On the basis of available literature (reviewed above) and knowledge itmay be concluded that the integration of titanium implants and bone andthe maintenance of this integration are prerequisites for the clinicallydocumented long-term function and high success rates. However, thekinetics of the process of osseointegration described above implies thatthe early phase of healing prior to adequate stability might beparticularly crucial in situations with an inferior bone quality andother negative host factors. Experimental studies on threaded titaniumimplants in more or less compromised local implant beds (previousirradiation of tissues or local inflammation and osteopenia) supportthis assumption (Sennerby and Thomsen 1993; Öhrnell et al., 1997).Further, studies in patients with rheumatoid arthritis have shown areduced mechanical capacity (decrease in torsional strength) of thebone-titanium unit in comparison with patients with osteoarthritis(Br{dot over (a)}nemark, 1996).

On the basis of the present results during the early phase of healing itis suggested that machined and electropolished c.p. titanium implantswith poly-crystalline, thick oxides and a microporous roughness on thesubmicron level may be interesting materials to be evaluated underclinical conditions. However, it is apparent that also an adequateremodelling of bone around the implants is required in order to promotea long-term stability. It is therefore of interest that in our long-term(1 year) study (paper IV) the experimental results showed that all fourtypes of threaded, titanium implants, irrespective of surfacemodification (machined, machined and anodized, electropolished,electropolished and anodized), had a high degree of bone-to-implantcontact and a high proportion of mature, lamellar bone within threads.Thus, implants with a similar chemical composition but with markeddifferences in oxide thickness, surface topography and roughness, becameequally well osseointegrated under long-term experimental conditions.

The examples given above have shown that it is possible to producetitanium implants with surface modifications which vary with respect tooxide thickness, composition, topography, roughness and microstructure.On the basis of results in thesis by Larsson (1997) it may be summarizedthat

in comparison with the merely electropolished implants (which had a verysmooth surface with a thin, non crystalline oxid) and machined implants,the implants which were surface modified with anodization aquired athicker oxid (180-200 nm), increased crystallinity and increasedroughness on the submicrometer scale.

A high degree of bone surrounding, and in contact with the implant, wasfound for all titanium implants, irrespective of surface modification.Taken together, the light microscopic, morphometric and ultrastructuralobservations indicate that the process of osseointegration is basicallysimilar for machined and surface modified titanium implants.

The results of the biological experiment show that a combination ofincreased oxide thickness, oxide crystallinity and roughness on thesubmicrometer scale are advantageous properties for the early boneresponse, particularly in comparison with thin, smooth non-crystallineoxide surfaces.

A high degree of bone-to-implant contact and a high proportion oflamellar bone within the threads of the implants are observed after oneyear, irrespective of surface modification (machined, machined plusanodized, electropolished and electropolished plus anodized). The latterresults indicate that the combination of surface properties (increasedoxide thickness, increased crystallinity and roughness on thesubmicrometer scale) of anodized implants have equal long-termbiological properties in bone as the clinically used machined titaniumimplants.

Taken together, our observations indicate that a titanium surface with acombination of surface properties (increased oxide thickness, increasedcrystallinity and roughness on the submicrometer scale), acquired in thepresent experiments by anodization, constitute an important element ofimplanted device.

TABLE I Summary of the results from surface characterization of theimplants. Oxide/ nitride Preparation thick- Rrms, Microstructure and(paper) Sterilization Contamination at % ness (nm) (nm) Adiff, % oxidecrystallinity Machined (I, III) steam sterilized 45-80 at % C 4 29 —Plastically deformed, (Ca, S, Si, P, Cl and Na) amorphous metal surface,non-crystalline oxide Electropolished (I, III) steam sterilized 55-90 at% C 4-5 2.7 — Polycrystalline metal sur- (Ca, S, Si, P, Cl, and Na)face, non-crystalline oxide Electropolished + anodized, steam sterilized55-70 at % C 21 1.5 — Polycrystalline metal sur- (I, III) (Ca, S, Si,and Cl) face, non-crystalline oxide Electropolished + anodized, steamsterilized 34-40 at % C 180 16 — Polycrystalline metal sur- (I, III) (Caand Cl) face. Partly rystalline oxide (anatase) Machined (II, IV) steamsterilized 34.4 at % C, 1.7 at % Cl, 3.5 3-5 30.3 10.8 Plasticallydeformed, amor- at % Na, (Ca, S, P, Si, and F) phous metal surface, non-crystalline oxide Machined + anodized, steam sterilized 33 at % C, 1.1at % Na, 180-200 40.8 18.0 Plastically deformed, amor- (II, IV) (Ca, S,P, Cl, and Si) phous metal surface, non- crystalline oxideElectropolished (II, IV) steam sterilized 26.9 at % C, (Ca, S, 3-5 2.90.5 Polycrystalline metal sur- Cl and Na) face, non-crystalline oxideElectropolished + anodized, steam sterilized 25.2 at % C 180-200 32.323.3 Polycrystalline metal sur- (II, IV) (S, Ci and Na) 2.7 0.6 facePartly crystalline oxide (smooth) (smooth) 116.7 88.0 (rough) (rough)Machined (V) γ-irradiated 23 at % C (Ca, Si, ≈3 26.3 13.1 Plasticallydeformed, amor- S and Cl) phous metal surface, non- crystalline oxideGlow discharge cleaned γ-irradiated 12 at % C (S) ≈2 10.2 0.78Polycrystalline metal sur- and thermally oxidized (V) face,non-crystalline oxide Glow discharge cleaned γ-irradiated 10 at % 0 andC (Si) TiN ≈ 3 25.2 8.63 Polycrystalline metal sur- and nitrided (V)face, non-crystalline oxide Hydrogen peroxide treated — 12 at % C (Ba,Cl and Zn) 7 25.6 20.5 Plastically deformed, amor- (V) phous metalsurface, Non- crystalline oxide

TABLE II The mean value for the three measurements (drops on one disc)are presented. The value for each drop (mean for right and left side ofthe drop) is presented within parentheses. H₂O H₂O CH₂I₂ CH₂I₂ advancingreceding advancing receding (contact (contact (contact (contactPreparation angles) angles) angles) angles) Machined 41.2 — 47 42 (37,44.5, (18, <10, (43, 46, 53) (39.5, 40.5, 42) 13) 45.5) Electropolished39.5 — 35 32 (38.5, 37.5, (12.5, 14.5, (37.5, 32, (31, 30.5, 42.5) <10)36.5) 33.5) Electropolished 22 <10 42.5 32 and (23, 22, (<10, <10,(45.5, 38.5, (35, 32.5, anodized 20.5) <10) 43.5) 29)

TABLE III The surface energy for the different samples. Surface energyPolar Dispersion Preparation dyne/cm component component Machined 58.133.6 24.4 Electropolished 61.3 32.2 29.1 Electropolished 68.6 42.8 25.8and anodized

What is claimed is:
 1. An implant element for permanent anchorage in bone tissue said element having at least one surface intended to face the tissue, wherein said surface comprises titanium, wherein said titanium surface is modified by hydrogen peroxide oxidation to acquire a titanium oxide coating that has a carbon content from 10 to 15 atom percent.
 2. An implant element according to claim 1, wherein the surface oxide crystallinity is altered to a polycrystalline structure.
 3. An implant element according to claim 2, wherein the ratio between the smooth and rough areas is larger than
 1. 4. An implant element according to claim 3 wherein the ratio between the smooth and rough areas is about
 3. 5. An implant element according to claim 2, wherein the rough areas are between 10×10 μm and 100×100 μm.
 6. An implant element according to claim 1, wherein the modified surface is a heterogenous surface with irregularly distributed smooth and rough areas.
 7. An implant element according to claim 1 wherein said titanium oxide coating has a thickness from 2 to 7 nm, as measured by Auger electron spectroscopy depth profile analysis.
 8. An implant element according to claim 1 wherein the carbon content is about 12 atom percent. 